Publications and other reference materials referred to herein are incorporated herein by this reference. The following description of the background of the invention is intended to aid in the understanding of the invention, but is not admitted to describe or constitute prior art to the invention.
The determination of the presence or amount of antigenic substances is commonly performed by receptor/immunoassay. Receptor/immunoassay techniques are based on the binding of the substance being assayed (the "target analyte") and a receptor for the target analyte. Either the target analyte or the receptor may be labeled to permit detection. Various labels have been employed for use in immunoassays, including radioisotopes, enzymes and fluorescent compounds. Many different types of immunoassays are known in the art, including competitive inhibition assays, sequential addition assays, direct "sandwich" assays, radioallergosorbent assays, radioimmunosorbent assays and enzyme-linked immunosorbent assays.
The basic reaction underlying most immunoassays is the binding of certain substance, termed the "ligand" or "analyte", by a characteristic protein (receptor) to form a macromolecular complex. These binding processes are reversible reactions, and the extent of complex formation for particular analyte and receptor concentrations is regulated by an equilibrium constant according to the law of mass action. Thus, at equilibrium, some of the analyte always exists unbound (free).
In a competitive inhibition immunoassay, the unknown quantity of target analyte in the sample competes with a known amount of labeled target analyte for a limited number of receptor binding sites. The reagents usually consist of a labeled target analyte, such as an antigen, and a solid phase coupled receptor, such as an antibody. The antigen to be assayed competes with the labeled antigen for binding sites on the coupled antibodies. The concentration of target analyte present in the sample can be determined by measuring the amount of labeled target analyte--either "free" or "bound." This is an indirect assay method where the amount of labeled antigen bound to the antibodies is inversely correlated with the amount of antigen in the test solution. Thus, low concentrations of target analyte in the sample will result in low concentrations of "free" labeled target analyte and high concentrations of "bound" labeled target analyte, and vice versa. The amount of "free" or "bound" labeled target analyte is measured using a suitable detector. Quantitative determinations are made by comparing the measure of labeled target analyte with that obtained for calibrated samples containing known quantities of the target analyte. This method has been applied to the assay of a great number of different polypeptide hormones, enzymes and immunoglobulins. This method may also be used as a total liquid system.
It is apparent to those skilled in the art that it is not absolutely necessary that the labeled analyte be identical to the unlabeled target analyte. If there is a difference between the two, for example, if the labeled analyte is an analog of the target analyte, the reaction between labeled and unlabeled analytes may be considered to be competitive for the receptor binding sites; and the reaction will still provide quantitative answers, providing the difference in affinity of the analytes is not too great. Whether or not true competition occurs in a system consisting of labeled analyte, unlabeled analyte, and receptor depends on the nature of the labeled analyte and the specificity of the receptor.
In sequential addition assays, the reagents used are the same as in the competitive inhibition assay described above. However, instead of incubating them at the same time, the unlabeled antigen is first incubated with the antibody, then the labeled antigen is added.
Direct immunoassay systems are also known in the art. Such assays, also termed "immunometric" assays, employ a labeled receptor (antibody) rather than a labeled analyte (antigen). In these assays the amount of labeled receptor associated with the complex is proportioned to the amount of analyte in the sample. Immunometric assays are well-suited to the detection of antigenic substances which are able to complex with two or more antibodies at the same time. In such "two-site" or "sandwich" assays, the antigenic substance has two antibodies bound to its surface at different locations. In a typical "forward" sandwich assay, an antibody bound to a solid phase is first contacted with the sample being tested to form a solid phase antibody:antigen complex. After incubation, the solid support is washed to remove the residual sample, including unreacted antigen, if any. The complex is then reacted with a solution containing a known amount of labeled antibody. After a second incubation to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed to remove unreacted labeled antibody. The assay can be used as a simple "yes/no" assay to determine whether the antigen is present. Quantitative determinations can be made by comparing the measure of labeled antibody with that of calibrated samples containing known quantities of antigen. "Simultaneous" and "reverse" sandwich assays are also known in the art. A simultaneous assay involves a single incubation step, both the labeled and unlabeled antibodies being added at the same time. A reverse assay involves the addition of labeled antibody followed by addition of unlabeled antibody bound to a suitable solid support. The sandwich technique can also be used to assay antibodies rather than antigens. Such an assay uses as a first receptor an antigen coupled to a solid phase. The antibodies being tested are first bound to the solid phase-coupled antigen. The solid phase is then washed, and then labeled anti-antibody (second receptor) is added.
The radioallergosorbent technique (RAST) is a method for the determination of antigen-specific IgE. The method uses a solid phase coupled antigen and an immunoabsorbent purified antibody labeled with a radioactive isotope. The method is used to detect reaginic antibodies against various antigens which elicit allergic reactions (allergens). The reaginic antibodies react with allergen bound to a solid matrix. After washing of the solid phase, the allergen-bound reaginic antibodies are detected by their ability to bind labeled antibodies against IgE. A variant of RAST can be used for the determination of allergens. The allergen to be tested is incubated with the reaginic antibody. The mixture is then tested with RAST using the same allergen coupled to the solid matrix. The allergen in solution reacts with the reaginic antibodies and thus inhibits the binding of these antibodies to the solid phase-coupled allergen.
Another assay method for the determination of IgE is the radioimmunosorbent technique ("RIST"). In this method, the solid support is sensitized with anti-IgE and increasing amounts of labeled IgE are added to determine the maximum amount of IgE that can bind. A quantity of labeled IgE equivalent to approximately 80% of the plateau binding is chosen. In the test experiments, this amount of labeled IgE is mixed with the serum containing the IgE to be tested. The test IgE competes with the labeled IgE. The more IgE present in the test serum the less the amount of labeled IgE that binds. Thus, by producing a standard curve the amount of IgE in a sample can be determined.
The above immunoassay methods can be applied to the assay of many different biologically active substances. Among such substances are haptens, hormones, gamma globulin, allergens, viruses, virus subunits, bacteria, toxins such as those associated with tetanus and animal venom, and many drugs. Similar techniques can be used in non-immunological systems with, for example, specific binding proteins.
Although some of the immunoassay methods described above utilize radioactive labels, those skilled in the art will appreciate that the assays can be adapted to use an alternate label, for example, a fluorophore.
If the properties of the label are not altered by binding, for example, as in a radioimmunoassay, a separation step is required to separate "free" from "bound" labeled target analyte. Such assays, which require a separation step, are called "heterogeneous" assays. If some particular property of the label is altered in some way when it is bound, no separation step is required, and the immunoassay is termed "homogeneous."
The measurement of target analytes in biological fluids, such as serum, plasma and whole blood, requires immunoassay methods which are both specific and sensitive. Both the specificity and sensitivity of an immunoassay depend on the characteristics of the binding interaction between the target analyte and the receptor involved. For example, the reaction must be specific for the analyte to be measured and the receptor used should not bind to any other structurally related compounds. In addition, by choosing a receptor with a high affinity for the target analyte, the sensitivity can be increased.
The label used to monitor the assay affects the sensitivity of an immunoassay. Labels currently used for immunoassay of target analytes in biological fluids include radioisotopes (radioimmunoassay, RIA), enzymes (enzyme immunoassay, EIA), fluorescent labels (fluorescence immunoassay, FIA), and chemiluminescent labels (chemiluminescent immunoassay, CIA).
RIAs are sufficiently sensitive for use in detection in low concentrations of analytes because of their low background. They are disadvantageous in that they are heterogeneous, thus requiring a separation step before measurement of the bound and/or free portions of labeled target analyte. RIAs involve the inconvenience and hazards associated with the handling and disposing of radioisotopes. In addition, they are labor intensive and have a short shelf life due to the half-lives of radiolabels and to chemical damage produced by the emitted radiation.
EIAs have the advantage of increased signal over background, longer shelf life, lack of radiation hazards, and homogeneity. They are disadvantageous in that, because they involve enzyme kinetic reactions, they are affected by the time of the kinetic measurements, as well as by variations in temperature, pH and ionic strength. The temperature of the enzyme incubation is particularly critical, and variations of more than 0.5.degree. C. can significantly affect assay results. Thus, drifts in standard curve may result from temperature fluctuation and inconsistencies in sample handling. Enzyme activity may also be affected by constituents in biological samples, such as plasma ant constituents. See generally Strong, J. E. and Altman, R. E., "Enzyme Immunoassay: Application to Therapeutic Drug Measurement," in P. Moyer et al., Applied Therapeutic Drug Monitoring, American Association of Clinical Chemistry (1984).
Chemiluminescent immunoassays (CIAs) offer a fairly high degree of sensitivity (picomole per liter range) but lack specificity in some instances. CIAs are disadvantageous because they are heterogeneous, require expensive reagents, and are expensive to automate. See generally Boeckx, R. L., "Luminescence: A New Analytical Tool for Therapeutic Drug Monitoring," in P. Moyer et al., Applied Therapeutic Drug Monitoring, American Association of Clinical Chemistry (1984).
FIAs use fluorescent molecules as labels. Fluorescent molecules (fluorophores) are molecules which absorb light at one wavelength and emit light at another wavelength. See Burd, J. F., "Fluoroimmunoassay--Application to Therapeutic Drug Measurement," in P. Moyer et al., Applied Therapeutic Drug Monitoring, American Association of Clinical Chemistry (1984). Typically, an excitation pulse of radiation is directed onto or into a sample, followed by fluorescence of the sample, and the detection of the fluorescence radiation.
FIAs may be either heterogeneous or homogeneous. As noted above, homogeneous assays are usually simpler to perform and are thus, more amenable to automation. However, previously known homogeneous FIAs are less sensitive than heterogeneous FIAs because high background can limit sensitivity. The heterogeneous FIA procedures can detect smaller amounts of analyte than present homogenous FIAs, but only because the separation and washing steps in the assays serve to eliminate background interference from biological substances. In solid phase fluorescent assays the solid support can limit sensitivity at the wavelengths of presently used fluors. In many cases the support itself will fluoresce at wavelengths of commonly used fluors such as fluorescein. FIAs also offer the advantage of using stable reagents.
Another assay method uses enzyme-enhanced fluorescence technology which combines microparticle capture and antigen-antibody reaction with an enzyme rate reaction using a fluorescent enzyme substrate. The rate reaction is monitored by steady state fluorometric measurement. In an enzyme-enhanced fluorescence assay, the analyte in question is "captured" by an antibody bound to a solid phase and the solid phase is washed. An enzyme is then bound to the captured analyte using an enzyme-anti analyte conjugate. Excess reactants are washed away and the amount of enzyme is measured by the addition of a non-fluorescent substrate. As the enzymatic reaction proceeds, the non-fluorescent substrate is converted to the fluorescent product. For example, an alkaline phosphatase-labeled antibody can be used to catalyze the hydrolysis of 4-methylumbelliferyl phosphate substrate to the fluorescent product methylumbelliferone. Thus, the rate at which the fluorescent product is generated is directly proportional to the concentration of analyte in the test solution. Enzyme-enhanced fluorescence assays, like EIAs, have the disadvantages associated with enzymes.
As discussed above, fluorescence is a phenomenon exhibited by certain substances, which causes them to emit light, usually in the visible range, when radiated by another light source. This is not reflection, but creation of new light. Current commercially available assay methods use fluorescein, which emits green light when radiated by a light source containing blue light.
In addition to fluorescing, fluorescein (and other fluorophores) emit polarized light. That is, the light emitted has the same direction of polarization as the incident polarized light, if the fluorescein molecule is held fixed with its transition moment parallel to the electric field of the excitation. The amount of polarization in the emission can be defined in terms of the intensity of the horizontally and vertically polarized light, as follows: EQU P=(Iv-Ih)/(Iv+Ih) (1)
where
Iv=intensity of vertically polarized emission PA1 Ih=intensity of horizontally polarized emission PA1 t=fluorescence lifetime, a constant PA1 r=rotational relaxation time PA1 R=gas constant PA1 T=temperature, .degree.K. PA1 n=solution viscosity PA1 V=volume of molecule PA1 (a) contacting a sample suspected of containing a target analyte with a first receptor capable of specifically recognizing said target analyte to form a complex of said target analyte and said first receptor, said first receptor being labeled with a fluorescent probe which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilized polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure; PA1 (b) contacting the complex with a second receptor capable of specifically recognizing said target analyte to, said second receptor being bound to a solid carrier, to form a complex of said first labeled receptor, said target analyte and said second receptor bound to said solid carrier; and PA1 (c) measuring either the amount of labeled first receptor associated with said solid carrier or the amount of unreacted labeled first receptor. PA1 (a) simultaneously contacting a sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing said target analyte, said first receptor being labeled with a fluorescent probe which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure, and said second receptor being bound to a solid carrier, to form a complex of said first receptor, said target analyte, and said second receptor; and PA1 (b) measuring either the amount of labeled first receptor associated with said solid carrier or the amount of unreacted labeled first receptor.
The maximum, or limiting value of polarization, for fixed, randomly oriented molecules is 0.5 (Po).
A second equation (the Perrin equation) defines polarization in terms of physical parameters and Po: EQU 1/P-1/3=(1/Po-1/3)(1+3t/r) (2)
where
Rotation relaxation is further defined for spherical molecules as EQU r=3nV/RT (3)
where
The rotational relaxation time is a measure of the rate at which a molecule will rotate when free in a solution. Note that the rotational relaxation time will typically be dependent primarily on the molecular volume and shape, since solution viscosity and temperature will be essentially constant in a normal assay. Thus, rotational relaxation time, and consequently, polarization, are affected only by the hydrodynamic properties of the molecule. The smaller a molecule is, the smaller its rotational relaxation time, and the faster it rotates (e.g., r=1 nsec for fluorescein, 100 nsec for large antibody complexes). For a constant, small, fluorescence lifetime (4 nsec for fluorescein), a small molecule retains little of the original polarization when irradiated by polarized light, because the molecule rotates rapidly and then fluoresces. On the other hand, a large molecule rotates slowly and for the same fluorescence lifetime, still retains a large degree of the original polarization when it fluoresces.
This dependency of polarization on molecular size can be used to determine the presence or amount of drug.
Using a fluorescent polarizing probe in a competitive binding immunoassay provides a type of FIA called a fluorescence polarization immunoassay (FPIA). In this type of assay, the smaller the molecule is, the smaller its rotational relaxation time and the faster it rotates. Typically, antibody molecules are much larger than drug or drug-probe molecules. For example, r=1 nsec for fluorescein and 57 nsec for gamma globulin.
When there is a large amount of drug present, there are very few binding sites available for the drug-probe. As a result, most of the probe (fluorescein) is in the form of small drug-probe molecules. As these molecules rotate randomly and rapidly, a low polarization value results. When there is a small amount of drug present, much of the drug-probe is bound to the large antibody molecules. These molecules rotate slowly, so the emitted light will be highly polarized.
The relationship between polarization and drug concentration can be determined by creating a standard, or calibration, curve. This is done by running an assay using a range of known drug concentrations, from the lowest to highest expected concentrations, and plotting the resulting values of polarization. Thereafter, for a given value of polarization, the drug concentration can be determined from the standard curve.
One advantage of the polarization technique is the elimination of a step to separate unbound probe. Although the unbound tracer is not physically eliminated from the samples in FPIA, its contribution is readily assessed by the polarization.
Another advantage in the FPIA technique is lack of dependence on intensity. In equation (1) above for calculating polarization using intensity, the ratio makes the polarization value unitless, or independent of variations in the intensity. Unlike most assays using a light measurement, in which it is the intensity of the light that is correlated to drug concentration (so any variations in source light intensity will directly affect the sensitivity of the assay), the sensitivity of FPIAs is independent of intensity variations. Conventional FPIAs require separate measurements of both blank and sample.
Theoretically, fluorometry is capable of being the most sensitive of all analytic tools as it is possible to detect single photon events. A problem which has plagued fluorescence immunoassays has been discriminating the fluorescent signal of interest from background radiation. The intensity of signal from background radiation may be up to 10,000 times larger than the intensity of the fluorescent signal of interest.
The problem of background detection is particularly pronounced in assay of biological samples. Many of the current fluorescence assays use the fluorescent molecule, fluorescein. Fluorescein has an excitation maximum of 493 nm, and there are numerous substances in biological fluids with overlapping excitation and emission similar to fluorescein. For example, in the analysis of blood plasma, the presence of a naturally occurring fluorescable material, biliverdin, causes substantial background radiation. Such compounds are highly fluorescent and contribute significant background signals which interfere with the label's signal, thus limiting the sensitivity of assays using fluorescein labels.
Earlier attempts to overcome the problem of background radiation have met with limited success. One technique for overcoming the problem involves discriminating against background radiation on the basis of wavelength. Filters have been used to reject detected radiation at all but a narrowly defined wavelength band. This technique has been less than successful principally because the background radiation may also be at the same wavelength as the desired fluorescence signal, accordingly, still be passed through the filter and detected.
It has been recognized that for analysis of biological fluids, it would be desirable to use a dye or label which is excitable at radiations of wavelengths of greater than background radiation. However, even though the background fluorescence of serum falls off at wavelengths approaching 600 nm, significant decrease does not occur until 650 nm or greater. Previous attempts to create dyes of such wavelengths have been unsuccessful. See, e.g., Rotenberg, H. and Margarfit, R., Biochem. Journal 2:197 (1985); and D. J. R. Laurence, Biochem. Journal 51:168 (1952).
A second technique attempting to discriminate the desired fluorescent signal from the background is the so called time gating approach. Here, the fluorescent signal is observed in a short time window after the excitation. The time window may be varied both in its length and in its starting time. Through the use of the variable time window, the detected radiation may be observed at the maximal time for detection sensitivity. Historically, this technique has used a fluorophore of very long decay time (such as 1,000 nanoseconds) to allow the background fluorescence to substantially decay before detection of the fluorescent signal of interest. Generally however, long decay time fluorophores require longer times for overall analysis. Due to the long decay time, the light source cannot be pulsed rapidly to collect data, thus requiring additional time for final analysis.
Historically, there have been two excitation pulse formats for transient state fluorescent analysis. One format utilizes a single, relatively high power pulse which excites the fluorophore. The transient state is typically monitored by a high speed photomultiplier tube by monitoring the analog signal representative of current as a function of time. Single pulse systems require sufficiently high power to excite a large number of fluorescent molecules to make detection reliable. The other principal format for transient state fluorescent analysis is a digital format which utilizes repetitive excitation pulses. Ordinarily, pulses of relatively short, typically nanosecond duration, light with power in the microwatt range are repetitively supplied to the sample at rates varying from 1 to 10,000 Hz. Ordinarily, the excitation source is a lamp, such as a Xenon-lamp. Frequently, the decay curve is measured digitally by determining the time to receipt of a photon. One commercially available system uses repetitive pulses (such as at 5,000 Hz) and pulses the photomultiplier tube at increasingly longer times after the flash in order to obtain a time dependent intensity signal. Detection in such systems has proved to be very time consuming and insensitive. A single analysis can take on the order of one hour, even at relatively high fluorescable dye concentrations (e.g., 10.sup.-6 M).
Recently, significant advances have been made in the area of fluorescable dyes. In one aspect, dyes being excitable by longer wavelength radiation, such as in the red and infrared wavelengths, are now available. These dyes are described in two commonly assigned patent applications: Arrhenius, U.S. patent application Ser. No. 701,449, filed May 15, 1991, entitled, "Fluorescent Marker Components and Fluorescent Probes," (which is a continuation-in-part of U.S. patent application Ser. No. 523,601, filed May 15, 1990), and Dandliker and Hsu, U.S. patent application Ser. No. 701,465, filed May 15, 1991, entitled "Fluorescent Dyes Free of Aggregation and Serum Binding" (which is a continuation-in-part of U.S. patent application Ser. No. 524,212, filed May 15, 1990). These applications are incorporated herein by this reference.
Further significant advancements have been made in increasing sensitivity through data collection and analysis techniques. As disclosed in Dandliker et al., U.S. Pat. No. 4,877,965, entitled "Fluorometer," which is incorporated herein by this reference, time gating techniques are used in conjunction with data collection and analysis techniques to obtain an improved signal relative to the background. Generally, the '965 Patent considers the detected intensity as a function of time to be composed of signals from various sources, including the desired signal source, and various undesired background sources. Optimization of the desired signal is achieved through data collection and analysis techniques.
Further significant advancements have been made in the ability to measure relevant materials in immunoassays. For example, using the technique described in Dandliker, et al., U.S. patent application Ser. No. 490,770, filed Mar. 6, 1990, entitled "Transient State Luminescence Assays," (which is a continuation-in-part of U.S. patent application Ser. No. 365,420, filed Jun. 13, 1989) incorporated herein by this reference, allows the bound and free form of materials in a homogeneous assay to be determined. Generally, the technique requires measurement of the time-dependent decay of the intensity of parallel and perpendicular polarization components. By measuring the time-dependent decay of various polarization states, it is possible to determine the bound and free forms of materials such as haptens, peptides, or small proteins in a homogeneous analysis format. Significantly, no separation of the bound and free materials is required.
Despite the significant and promising improvements made in the field of fluorescable dyes, and in the data analysis aspects, the actual methods and apparatus for achieving and detecting fluorescence have heretofore remained relatively unchanged. Utilizing even the most sensitive and best equipment, analysis can take an hour or more, even at high concentrations of materials. When fluorometry is used for immunoassay in a clinical context, time for analysis and proper diagnosis can be absolutely critical. Patient survival can depend on accurate, timely analysis. Additionally, rapid testing would permit retests of patients without having them wait significant periods of time, resulting in more rapid and accurate diagnosis. As to sensitivity, it is extremely desirable to be able to detect minute amounts of fluorescable material. However, as the amount of fluorescable material in a sample decreases, the ratio of background to signal increases. Further, since the time for analysis depends on the amount of fluorescent radiation received from the detector, low concentrations generally require substantially more time to analyze.
Heretofore, the time required for analysis has been prohibitively high. Known methods and apparatus for FIAs have failed to provide rapid and accurate diagnosis and analysis of samples. This has been so despite the clear and known desirability of the use of FIAs. For example, the drug digoxin, which is used to treat congestive heart failure, has a narrow therapeutic range (i.e., serum levels of 0.5 to 2.5 ng/ml and is generally toxic at concentrations greater than 2.1 ng/ml. Present assays using fluorescence-based methodologies require an extracting process to remove interfering substances, such as proteins, in order to detect digoxin at its therapeutic levels. This additional extraction step increases the time, cost and equipment needed to perform the assay.
From the above discussion it can be seen that, although many different types of immunoassays currently exist, none is satisfactory for measuring small quantities of target analytes in biological fluids such as serum, plasma and, especially, whole blood. Accordingly, an object of the present invention is to provide improved processes for assay of antigenic substances. More specifically, the present invention provides fluorescence assays which allow the detection of low levels of antigenic substances in biological samples such as serum, plasma and whole blood. The present invention also provides homogeneous fluorescence assays which allow rapid and accurate determination of low levels of antigenic substances in biological samples.